The reversible covalent modification of proteins comprises a major mechanism of cellular regulation. Interconversion of a phosphorylatable protein is catalyzed by a protein kinase (PK) and a phosphoprotein phosphatase (PT). Because of the central regulatory role of cyclic phosphorylation/dephosphorylation cascade systems, it is important to understand qualitatively and quantitatively the characteristics both of the total system and of the enzymes involved therein. To this end, a model in vitro phosphorylation/dephosphorylation cascade was developed in which the two converter enzymes were bovine cardiac cAMP-dependent PK (type II) and a type-2 phosphoprotein PT (MW = 38,000), and the interconvertible substrate was a nanopeptide analogous to the site phosphorylated in rat liver pyruvate kinase. In an attempt to determine conclusively the stoichiometry and mechanism of activation of the cAMP-dependent PK by cAMP, activation profiles were performed over a 10-fold range of kinase concentrations. The data were analyzed by computer curve-fitting to kinetic equations derived to define the reaction. These studies led to a plausible description of the steps involved in cAMP activation of the kinase, thereby enabling a precise analysis of the involvement of this enzyme in the cyclic cascade. Extensive experiments on the steady state levels of phosphorylation of the nanopeptide and on the ATP flux in the cascade were carried out at various levels of the converter enzymes and effectors (cAMP and Pi). The experimental data were delineated completely by deriving appropriate kinetic and steady state equations based upon theoretical predictions for the behavior of the cyclic cascade and including the kinetic information determine for the individual converter enzymes (PK and PT). In a separate collaboration, phosphatases in Erhlich ascites tumor cell extracts were identified and partially purified using specific PT substrates to distinguish between different PT activities. In addition, heat stable PT inhibitors in these extracts were analyzed because of the importance of identifying in vivo regulators. Of particular interest was an inhibitor for the type-2 phosphatase. The inhibitory activity was isolated and partially characterized.